Cell-permeable small molecules can rapidly perturb the function of their targets and are therefore powerful tools to dissect dynamic cellular processes. However, such modulators are not available for most of the proteins involved in essential processes, and many of the ones that are available are nonspecific. The only known small molecules that specifically affect the mitotic machinery target tubulin (E. Hamel, Med. Res. Rev. 16, 207 (1996)), a subunit of the microtubules in the mitotic spindle.
One class of proteins involved in the assembly and maintenance of the mitotic spindle is the family of mitotic kinesins, a subset of the kinesin superfamily. This superfamily contains over 100 proteins, whose other functions include organelle transport and membrane organization (R. D. Vale and R. J. Fletterick, Annu. Rev. Cell Dev. Biol. 13, 745 (1997)). The first evidence that mitotic kinesins are important in establishing spindle bipolarity came from genetic studies: temperature-sensitive mutants in the BimC family of kinesins do not form bipolar spindles at the restrictive temperature (A. P. Enos and N. R. Morris, Cell 60, 1019 (1990); I. Hagan and M. Yanagida, Nature 356, 74 (1992); M. A. Hoyt et al., J. Cell Biol. 118, 109 (1992)). Inhibition of the BimC kinesin Eg5 with Eg5-specific antibodies also induced monoasters similar to those observed after treatment with monastrol (A. Blangy et al., Cell 83, 1159 (1995); K. E. Sawin et al., Nature 359, 540 (1992)). Like other kinesins, Eg5 can drive the movement of microtubules in vitro (T. M. Kapoor and T. J. Mitchison, Proc. Natl. Acad. Sci. U.S.A. 96, 9106 (1999)).
Enzymes in the kinesin superfamily use the free energy of ATP hydrolysis to drive intracellular movement and influence cytoskeleton organization (R. D. Vale and R. J. Fletterick, Annu. Rev. Cell. Dev. Biol. 13, 745-777 (1997)). More than 90 members of this family are known. Historically, kinesins have been proposed to move cellular cargo along polar microtubule tracks. More recently it has been shown that these ATPases can modulate dynamics of the underlying microtubule network (A. Desai et al., Cell 96, 69-78 (1999)), couple movement of cargo to the microtubule polymerization or depolymerization (K. W. Wood et al., Cell 91, 357-366 (1997)), and crosslink microtubules in dynamic structures (D. J. Sharp et al., J. Cell Biol. 144, 125-138 (1999)). Kinesins thus play central roles in mitotic and meiotic spindle formation, chromosome alignment and separation, axonal transport, endocytosis, secretion, and membrane trafficking. The cargo associated with these motor proteins includes intracellular vesicles, organelles, chromosomes, kinetochores, intermediate filaments, microtubules, and even other motors (reviewed in C. E. Walczak and T. J. Mitchison, Cell 85, 943-946 (1996); and N. Hirokawa, Science 279, 519-526 (1998)).
For many of these processes, more than one kinesin is implicated, and the specific cargo associated with a given motor protein has been difficult to establish. For example, conventional kinesin (R. D. Vale et al., Cell 42, 39-50 (1985)) (the founding member of the family) is one of a subset of kinesins involved in organelle transport in mammalian cells. This group includes KIF1, KIF2, KIFC2/C3, and KIF4; and more recently, 18 new murine KIFs have been reported, many of which may functionally overlap with the transport kinesins (reviewed in N. Hirokawa, Science 279, 519-526 (1998)). It thus has been difficult to tie down the in vivo function(s) of conventional kinesin. Experiments using antisense techniques and microinjection of inhibitory antibodies have been further complicated by recent observations of efficient endoplasmic reticulum to Golgi transport in the absence of microtubules, albeit under restricted conditions (reviewed in G. S. Bloom and L. S. Goldstein, J. Cell Biol. 140, 1277-1280 (1998)). Similar problems have been encountered in dissecting the function of kinesins in mitosis. Extensive genetic analysis of motors in Saccharomyces cerevisiae has linked all but one of the six kinesins to spindle function. None of these five motors are individually required for the viability of yeast, implying that more than one motor is associated with essential aspects of spindle movement (W. S. Saunders and M. A. Hoyt, Cell 70, 451-458 (1992); M. A. Hoyt et al., Proc. Natl. Acad Sci USA 94, 12747-12748 (1997)). Immunodepletion and add-back approaches in Xenopus extract spindle assembly assays have provided similarly ambiguous data (C. E. Walczak et al., Curr. Biol. 8, 903-913 (1998)).
Small molecules that conditionally activate or inactivate a protein are valuable tools for analyzing cellular functions of proteins (D. T. Hung et al., Chem. Biol. 3, 623-639 (1996)). Their use provides an alternative to conventional biochemical and genetic approaches. However, to date there have been few reports of small molecules that can reversibly alter the function of motor proteins. Butanedione monoxime has been used to probe the role of myosin in cell movement (L. P. Cramer and T. J. Mitchison, J. Cell Biol. 131, 179-189 (1995)), but its specificity has been questioned (G. Steinberg and J. R. McIntosh, Eur. J. Cell Biol. 77, 284-293 (1998)). A natural product inhibitor of kinesin has been reported (R. Sakowicz et al., Science 280, 292-295 (1998)), but is thought not to be selective for different kinesins and thus is not useful for probing the role of one specific kinesin in a complex process. Hyman et al. (A. A. Hyman et al., Nature (London) 359, 533-536 (1992)) have used ATP analogs to distinguish between microtubule motility at kinetochores driven by a kinesin and a dynein, but again, this approach is unlikely to distinguish between different kinesins. Thus currently there is a lack of small molecule activators or inhibitors that are specific for one member of the kinesin family. Such an inhibitory molecule with specificity for a particular member of a kinesin class would be useful as an anti-mitotic and also as an anti-cancer, anti-tumorigenic compound.